In general, various physiological functions are regulated by the dynamic interactions between various bioactive molecules. If such interactions do not occur properly, problems arise to cause diseases. For example, proteins in vivo perform their functions by interaction with other proteins. Generally, two proteins having complementary structures interact with each other, and a bioactive compound interacts specifically with the specific portion of the three-dimensional protein structure. Generally, the interaction between two proteins strongly implies that they are functionally related. Furthermore, a bioactive compound interacting specifically with the specific portion of a disease-associated protein has potential as a therapeutic agent which can diagnose, prevent, treat or alleviate the disease by controlling the activity of the protein.
Accordingly, in the field of new drug development, there have been studies on various methods of detecting novel target proteins or screening bioactive molecules as drug candidates by detecting the interaction between a “bait” whose function and feature are known and a “prey” which is an interaction partner to be analyzed and detected. Thus, the identification and isolation of a novel target protein through the analysis of the interaction between bioactive molecules are considered as very important research projects for obtaining useful information about the activity, effectiveness and adverse effects of bioactive drugs. Additionally, target proteins promote the understanding of biological pathways and signal transduction systems and provide information on fundamental cellular regulation and disease mechanisms. Such information is a very powerful tool for developing new drugs, improving existing drugs and discovering the novel pharmaceutical use of existing drugs by analyzing and detecting bioactive compounds which interact with the target proteins.
Modern medicine faces the challenge of developing safer and more effective therapies against various human diseases. However, many drugs currently in use are prescribed by the biological effects in disease models without their target proteins and molecular targets (Burdine, L. et al., Chem. Biol. 11: 593, 2004). Bioactive natural products are an important source for drug development, but their modes of action are usually unknown (Clardy, J. et al., Nature 432:829, 2004). Elucidation of their physiological targets and molecular targets is essential for understanding their therapeutic and adverse effects, thereby enabling the development of improved second-generation therapeutics. Moreover, the discovery of novel targets of clinically proven compounds may suggest new therapeutic applications (Ashburn, T. T. et al., Drug Discov. 3:673, 2004).
In chemical and biological field employing cell-based screening, “target screening” is used to identify small molecules with a desired phenotype from large compound libraries (Strausberg, R. L. at al., Science 300:294, 2003; Stockwell, B. R. Nature 432:846, 2004). Despite the great benefits of such screening, this approach has been hampered by the daunting task of target identification. However, the development of such identification technology is very important in various bioscience fields, including genomics, proteomics and system biology, because effective detection of diverse intracellular molecular interactions, including protein (or small molecule)-protein, is essential for understanding dynamic biological processes and regulatory networks.
In the field of target screening, several technologies, including affinity chromatography (Phizicky, E. M. et al., Microbiol. Rev. 59:94, 1995; Mendelsohn, A. R. et al., Science 284:1948, 1999), protein-small molecule microarray, phage display (Sche, P. P. et al., Chem. Biol. 6:707, 1999), yeast two-/three-hybrid assay (Licitra, E. J. et al., Proc. Natl. Acad. Sci. USA 93:12817, 1996), expression profiling, and parallel analysis (Zheng, O. et al., Chem. Biol. 11: 609, 2004) of yeast strains with heterologous deletions, have been utilized to analyze interactions between bioactive molecules.
However, such technologies all suffer from diverse problems, including high background, false positives, low sensitivity, inappropriate folding after protein expression, indirectness, lack of modification after protein expression, or limited target accessibility including cellular compatibility. In addition, the use of artificial experimental milieu, such as in vitro binding conditions or non-mammalian cells, sometimes causes errors in experimental results.
Accordingly, it is most preferable to directly examine the interaction between bioactive molecules in a state in which high sensitivity and selectivity were considered in physiological or pharmaceutical terms. Thus, it is considered that it is very important to develop the above-described base technology in order to offer various advantages over the prior art.
First, by probing the interactions between physiologically or pharmaceutically relevant bioactive materials and molecules, misleading outcomes produced by an artificial experimental setting can be greatly diminished. Second, it is possible to directly translate the interaction between bioactive molecules into a clear readout signal, unlike indirect readout methods that are dependent on overall expression profiles or complex biological phenotypes. Thus, intrinsic false positives/negatives or error-prone deductions about bioactive molecules and molecular targets can be obviated. Third, it is possible to perform dynamic, single-cell analysis for the interactions between bioactive materials and molecules. Dynamic analysis of individual living cells provides an effective method which can analyze intracellular processes occurring non-simultaneously among heterogeneous cells, over a broad range in physiological and pharmaceutical terms.
Therefore, the above-described base technology can be used to detect a variety of biological interactions between bioactive materials and molecules (e.g., interaction between a bioactive small molecule and a protein) and protein modifications (e.g., phosphorylation) within living cells in a broad range of tissues and disease states, but have many limitations. Thus, the development of new base technology is required.
Accordingly, the present inventors disclosed a method of investigating and verifying the dynamic interaction between various bioactive materials by analyzing whether this dynamic interaction results in nano-assembly matrix formation or co-localization on a nano-assembly matrix, by imaging (Korean Patent Laid-Open Publication No. 2009-0018585). This method is a qualitative method capable of investigating and verifying interactions by imaging using a microscope or the like.
Meanwhile, various physiological (assembly) matrices are present as signalsome in cells and as “-some” or “complex” such as exosome in an extracellular environment in vivo. It is known that bioactive materials, including one or more relevant proteins, are present in such physiological assembly matrices, and thus specific physiological functions in cells or in vivo are effectively regulated. It was found that multi/poly-valent interactions play a very important role in most physiological regulations, like in efficient physiological regulation mediated by such matrices (Mammen, M. et al., Angew. Chem. Int. Ed. 37:2755, 1998; Kiessling, L. L. et al., Angew. Chem. Int. Ed. 45:2348, 2006). In other words, multi/poly-valent interactions mainly play an important role in the interactions between most bioactive materials, including proteins, compared to mono-valent interactions.
Accordingly, the present inventors have conducted additional studies based on the high-density characteristic of such physiological matrices, and as a result, have developed novel methods capable of performing analysis and detection in a quantitative manner and detection and labeling in a more effective manner, deviating from a quantitative analysis method based on visual imaging. When energy transfer and signal change, which effectively occur between label molecules displayed on a nano-assembly matrix at high density, are measured, interactions between materials can be qualitatively and quantitatively measured and detected by, for example, a method including fluorescence analysis or FRET (fluorescence resonance energy transfer). In addition, when the efficiency of interactions between detector materials is increased by displaying the materials on a nano-assembly matrix at high density, the interactions between the materials can be qualitatively and quantitatively measured and detected in a more sensitive manner. Thus, there are advantages in that labeling is easy, interactions between materials can be individually identified in an easier manner by the quantitative analysis of energy signals using a method including flow cytometry or FACS (fluorescence associated cell sorting), and the high throughput of detection and analysis can be increased. The present inventors have found that a specific target material that efficiently interacts with detector materials displayed on a nano-assembly matrix at high density can be sensitively detected and labeled on the basis of such energy transfer or signal change, thereby completing the present invention.
The information disclosed in the Background Art section is only for the enhancement of understanding of the background of the present invention, and therefore may not contain information that forms a prior art that would already be known to a person of ordinary skill in the art.